ABSTRACT: Angiogenesis is a dynamic process essential for primary tumor growth and metastases. New insights into the basic understanding of the biologic processes responsible for angiogenesis have led to the characterization of potential therapeutic targets. Several strategies for the development of antiangiogenic therapeutic modalities have been employed, including agents that (1) decrease the activity of specific angiogenic factors, (2) decrease the activity of endothelial survival factors, (3) increase the activity of naturally occurring antiangiogenic agents, or (4) indirectly downregulate angiogenic and survival factor activity. Because antiangiogenic therapy is unlikely to induce tumor regression, the criteria for efficacy must be evaluated by means other than the standard response criteria used to evaluate cytotoxic chemotherapy. Further, the redundancy of molecules responsible for the angiogenic process suggests it is unlikely that a single antiangiogenic agent will provide prolonged inhibition of angiogenesis. Nevertheless, the understanding of the basic principles that drive tumor angiogenesis will lead to the development of therapies that will likely prolong survival without the toxicity associated with standard chemotherapy. [ONCOLOGY 16(Suppl 4):14-22, 2002]

By definition, angiogenesis is the establishment of a
neovascular blood supply derived from preexisting blood vessels, whereas
vasculogenesis is the embryonic establishment of a blood supply from mesodermal
precursors such as angioblasts or hemangioblasts. Tumor angiogenesis more
accurately refers to a combination of angiogenesis and vasculogenesis in which
the main blood supply to a tumor is derived from preexisting blood vessels,
although circulating endothelial cell precursors may contribute to the growing
endothelial cell mass.

Numerous investigators have established the association of tumor angiogenesis
with metastasis.[1] Indeed, it is thought that tumor angiogenesis is essential
for the growth of both primary and metastatic tumors,[2,3] and provides both
nutrients and oxygen to the growing tumor mass. A neovascular blood supply is
also essential for increasing the chance that tumor cells will gain access to
the circulation and subsequently begin the process of forming metastases at
different sites. Once a tumor establishes an invasive phenotype in the organ of
metastasis, it must then establish its own neovascular blood supply in order to
grow.

This process, more complex than was previously thought, requires the
coordinated activities of multiple factors and cell types. For tumors to develop
a neovascular blood supply, tumor and host cells must secrete proangiogenic
factors that offset the activities of inhibitory angiogenic factors. In
addition, the newly derived tumor endothelium must respond to and survive in a
relatively caustic microenvironment; thus, endothelial cell-survival factors are
essential in the maintenance of this neovasculature. Nevertheless, because the
process of angiogenesis is regulated by redundant factors and pathways,
inhibition of any single pathway is unlikely to lead to prolonged response in
most patients with solid malignancies.

More than 1,700 papers were published on aspects of tumor angiogenesis in
2001. This field of research is closely scrutinized by scientists, clinicians,
patients, and the media. However, data from phase I and II antiangiogenic trials
have only been reported in abstract form; most of the data is too preliminary to
draw meaningful conclusions. Further, phase III trials, even if they have
reached their target accrual, are several years away from maturity with
appropriate follow-up. The published reports available on clinical trials have
thus far produced little more than information on the toxicity and tolerability
of angiogenesis inhibitors.

Given the complexity of angiogenesis, the basic biology of this process must
be better understood before effective antiangiogenic therapy can be developed.
Herein, we review recent advances in the basic understanding of angiogenesis and
the role of angiogenic factors in tumorigenesis. Further, we will discuss
overall strategies, expectations, and future directions of antiangiogenesis
therapy.

Under normal physiologic conditions, the activity of endogenous
pro-angiogenic factors equals that of antiangiogenic factors, leading to a
homeostatic balance that prevents the uncontrolled growth of tissues. Pathologic
angiogenesis occurs when the effect of stimulatory molecules outweighs the
effect of inhibitory molecules (Table 1).[4] Intensive study of the angiogenic
process led to the realization that this process involves more than simple
proliferation of endothelial cells. This process also requires endothelial cells
to divide, invade the basement membrane, migrate, and undergo differentiation
and capillary-tube formation (Figure 1).[4] This process is driven not only by
angiogenic molecules, but also by other factors, such as degradative enzymes,
that mediate the above processes. Interestingly, the processes of tumor
angiogenesis (as noted above) and the processes of tumor-cell invasion are very
similar.

Vascular Endothelial Growth Factor

The best characterized of the stimulatory angiogenic factors is vascular
endothelial growth factor (VEGF), which has also been associated with an
aggressive phenotype in numerous solid malignancies.[5-10] Vascular endothelial
growth factor is a 32- to 44-kDa protein secreted by nearly all cells.[4] At
least four isoforms of VEGF, derived from alternate splicing of the mRNA, have
been characterized.[4,11] The smaller isoforms, VEGF-121 and VEGF-165 (the
numbers denote the number of amino acids), are secreted from cells. The larger
isoforms, VEGF-189 and VEGF-205, are cell associated, and their functions are
currently being investigated.

One distinguishing factor of VEGF is its ability to induce vascular
permeability. In fact, this factor was originally named vascular permeability
factor (VPF) and was subsequently found to be homologous to VEGF.[12-14] The
extent of vascular permeability induced by VEGF is 50,000 times that of
histamine, which was historically the gold standard for induction of
permeability. This action by VEGF allows proteins to diffuse into the
interstitium and to form the lattice network onto which endothelial cells
migrate.

In the past, it was believed that receptors for VEGF were expressed
predominantly on endothelial cells. Recently, the VEGF receptors have also been
found on cells of neural origin, Kaposi’s sarcoma cells, hematopoietic
precursor cells, certain leukemias, and selected epithelial tumors.[15,16] The
current nomenclature for the three known VEGF receptors is VEGFR-1(Flt-1),
VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4). These tyrosine kinase receptors
require dimerization to induce intracellular signaling following specific ligand
binding. The receptors for VEGF may mediate distinct functions within the
endothelial cell. For example, VEGFR-1 may be important in migration, whereas
VEGFR-2 may be important in the induction of permeability, endothelial cell
proliferation, and survival. Neuropilin, a receptor involved in neuronal
guidance, has been identified as a coreceptor for VEGF-165 and may enhance
angiogenesis.

Recently, the angiopoietin family of ligands has been found to play an
important role in the homeostasis of the tumor vasculature. The angiopoietins
are proteins involved in angiogenesis that bind to the endothelial-cell-specific
tyrosine kinase receptor Tie-2. Angiopoietin-1 (Ang-1) acts as an agonist and is
involved in endothelial-cell differentiation and stabilization.[17] In contrast,
Ang-2 binds to Tie-2 and blocks the binding of Ang-1 to this receptor.[18,19]
This blockade leads to endothelial-cell destabilization and vascular
regression.[20]

Angiogeneis Hypotheses

It has been hypothesized that tumor angiogenesis involves the co-option of
preexisting blood vessels in addition to vascular regression and subsequent
neovascularization.[20] This theory suggests that tumors initially co-opt
existing blood vessels within an organ for their nutrient blood supply. Shortly
thereafter, the existing vasculature becomes destabilized, most likely through
the release of Ang-2 by endothelial cells. This loss of vascular integrity leads
to relative hypoxia within the tumor, which in turn leads to upregulation of
VEGF in the tumor cells. These events then lead to a robust angiogenic response.
At that stage, the newly developed endothelial cells require stabilization,
which is achieved through release of Ang-1 by endothelial cells and possibly
through continued response to VEGF. Thus, the process of angiogenesis depends on
the temporal coordination of factors that regulate pathways in the establishment
of stable conduits that provide a nutrient blood supply to the tumor.

In vitro, Ang-1 has been shown to be angiogenic, inducing tube formation of
endothelial cells growing on extracellular matrix components. However, recent in
vivo studies have demonstrated that Ang-1 may in fact be antiangiogenic. We have
shown that overexpression of Ang-1 in human colon cancer cells leads to
decreased angiogenesis and tumor growth, whereas overexpression of Ang-2 leads
to an increase in tumor growth and angiogenesis.[21] This finding is consistent
with immunohistochemical studies that demonstrate that colon cancers express
Ang-2 but do not express Ang-1. This suggests that the imbalance of Ang-2 over
Ang-1 may be an initiating factor in tumor angiogenesis. Others have also
confirmed the above findings in breast and gastric cancer tumor cells and cell
lines.[22,23]

These factors are known to be angiogenic in in vivo models but are not
specific for endothelial cells. However, as noted earlier, a single molecule or
family of molecules does not drive angiogenesis; rather it depends on the
cooperation and integration of various factors leading to endothelial cell
proliferation, migration, invasion, differentiation, and capillary-tube
formation. It has yet to be determined whether inhibiting the activity of a
single angiogenic factor will lead to vascular compromise of significant
duration. More likely, the redundancy in the angiogenic process will select for
other angiogenic factors when a specific angiogenic factor is targeted.